Mitigation of liquid–liquid phase separation of a monoclonal antibody by mutations of negative charges on the Fab surface

Abstract

Some monoclonal antibodies undergo liquid–liquid phase separation owing to self-attractive associations involving electrostatic and other soft interactions, thereby rendering monoclonal antibodies unsuitable as therapeutics. To mitigate the phase separation, formulation optimization is often performed. However, this is sometimes unsuccessful because of the limited time for the development of therapeutic antibodies. Thus, protein mutations with appropriate design are required. In this report, we describe a case study involving the design of mutants of negatively charged surface residues to reduce liquid–liquid phase separation propensity. Physicochemical analysis of the resulting mutants demonstrated the mutual correlation between the sign of second virial coefficient B 2 , the Fab dipole moment, and the reduction of liquid–liquid phase separation propensity. Moreover, both the magnitude and direction of the dipole moment appeared to be essential for liquid–liquid phase separation propensity, where electrostatic interaction was the dominant mechanism. These findings could contribute to a better design of mutants with reduced liquid–liquid phase separation propensity and improved drug-like biophysical properties.

Suppression of Electrostatic Mediated Antibody Liquid–Liquid Phase Separation by Charged and Noncharged Preferentially Excluded Excipients

“…For example, at pH 5, no LLPS was observed without the addition of the macromolecular crowding agent PEG-6000 (Figure ). Finally, a variant of mAb-B (mAb-B CM) containing three charge mutations in the variable regions (generated for purposes other than to mitigate LLPS) subtly delocalized these charge patches and this variant required roughly 10% (w/v) more PEG-6000 than mAb-B to induce the same degree of LLPS in identical solvent conditions (Figure ), similar to recent reports of other mAbs where disruption of charged patches in the variable regions were also shown to mitigate LLPS. ,, …”

“…Finally, a variant of mAb-B (mAb-B CM) containing three charge mutations in the variable regions (generated for purposes other than to mitigate LLPS) subtly delocalized these charge patches and this variant required roughly 10% (w/v) more PEG-6000 than mAb-B to induce the same degree of LLPS in identical solvent conditions (Figure 5), similar to recent reports of other mAbs where disruption of charged patches in the variable regions were also shown to mitigate LLPS. 29,45,47 Based on these results, we conclude that mAb-B LLPS is mediated through localized charge patches on the surface of the Fab and that the excipients studied in this report preferentially interact with these specific residues despite their net unfavorable interaction with the entire mAb measured by VPO. In support of this idea, we note that apparent transfer free energies have been experimentally determined at neutral pH for the amino acid side chains and a model unit of the peptide backbone from water into a number of different excipients.…”

“…Instead, mAb-B homology models displayed regions of localized surface charge, particularly in the variable regions of the Fab (Figure ). Although single amino acid residues can have dramatic impacts on protein biophysical properties including their state of association and conformational stability, recent studies have demonstrated that both the magnitude and direction of mAb dipole moments resulting from surface charge asymmetry correlate with PPIs, particularly at high protein concentrations, responsible for increased solution viscosity and LLPS. − Charge patch analysis of homology models derived from the variable domain sequences of 137 mAbs that have been in clinical trials revealed that only 4 had a greater sum of positive and negative charged solvent accessible surface areas in their variable domains than mAb-B. These areas seem most likely to be those responsible for attractive mAb-B PPIs given the marked decrease in LLPS following addition of as little as 10 mM KCl (Figure ).…”

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay

“…Another hallmark of low colloidal stability of HCPFs is reversible liquid–liquid phase separation during refrigerated storage. ,,, LLPS typically occurs shortly upon lowering the temperature, while crystallization and colloidal aggregation are often observed over long-term storage. The propensity of HCPFs to undergo LLPS is measured by the cloud-point temperature, T cloud,high c , upon cooling the protein samples.…”

Antibodies with Weakly Basic Isoelectric Points Minimize Trade-offs between Formulation and Physiological Colloidal Properties

“…Although it is desirable to identify antibodies with both low non-specific interactions and low self-association, a holistic analysis of previous studies of each individual property suggests that this may be particularly challenging. For example, strongly positively charged antibodies with high isoelectric points (pIs) have been shown to display low risk for high self-association, viscosity, and opalescence under standard formulation conditions. ,− However, the same type of strongly positively charged antibodies have also been shown to display high risk for non-specific interactions under physiological conditions (pH 7.4 and PBS) and fast antibody clearance in vivo. ,− Likewise, strongly negatively charged antibodies with low pIs have been shown to display high risk for high self-association, viscosity, and opalescence under standard formulation conditions, while the same antibodies display low-to-medium risk for non-specific interactions under physiological conditions and fast antibody clearance in vivo.…”